The ability of penicillin and other modern antibiotics to control infectious diseases has been steadily eroded by the emergence of multiple-antibiotic-resistant strains of bacteria. The CDC estimates that 90,000 Americans die from bacterial infections annually, with 70% of these infections involving antibiotic-resistant strains. Bacterial survival strictly depends on the functionality of the cytosolic enzyme MurA, which catalyzes the first committed step in the synthesis of the bacterial cell. It is the target of the naturally occurring, broad spectrum antibiotic fosfomycin. Fosfomycin, an epoxide, covalently attacks a critical cysteine-residue of MurA (Cys115). This residue-directed mode of action renders fosfomycin a relatively poor drug, because in pathogenic bacteria, such as Mycobacterium tuberculosis, Cys115 is replaced by an aspartate. In addition, an ever-increasing number of pathogenic bacteria have developed resistance to fosfomycin due to decreased transport of fosfomycin into the cell, and inactivation by a fosfomycin resistance protein (FosA). Thus, there is a critical need for the development of novel drugs targeting MurA by a different molecular mode of action. We have recently identified five new MurA inhibitors with unique scaffolds and IC50 values ranging from 2 mu M to 8 mu M by high-throughput screening (HTS) using a 50,000 compound library. The central goal of this proposal is to thoroughly evaluate the mode of action of these new lead structures on MurA. The specific aim, which integrates enzyme kinetics and protein crystallography, is to perform inhibition kinetics and determine the crystal structure of MurA bound with these inhibitors in order to resolve the structure-activity relationships in atomic detail. The long-term goal of this proposal is to provide a basis for the rational design of novel potent broad-spectrum antibacterial drugs that specifically target MurA.